Modified, hydroxy-substituted aromatic structures having cytoprotective activity

ABSTRACT

The present invention is directed to a process for conferring cytoprotection on a population of cells which comprises administering to that population of cells a compound comprising a hydroxy-substituted aromatic ring structure and a non-fused polycyclic, hydrophobic substituent attached thereto. In particular, the present invention is directed to such a process wherein the administered compound is phenolic, such as a steriod (e.g., estrogen), and has a non-fused polycyclic, hydrophobic substituent attached to the hydroxy-substituted A-ring thereof.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/971,423, filed Oct. 22, 2004, which is a divisional of U.S.application Ser. No. 10/007,450, filed Nov. 5, 2001, now U.S. Pat. No.6,844,456, which claims priority from U.S. provisional application Ser.No. 60/245,791, filed on Nov. 3, 2000. The entire contents of both ofthese applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention is generally directed to modified,hydroxy-substituted or hydroxy-bearing aromatic structures havingcytoprotective activity, as well as to a treatment process involving theadministration of an effective dosage thereof. More specifically, thepresent invention is directed to phenolic compounds or catecholiccompounds which have been modified by the attachment of a non-fusedpolycyclic, hydrophobic substituent. Such compounds have been found topossess enhanced cytoprotective activity, as compared to theirrespective analogs which do not contain such a substituent. Thisactivity may be conferred to a population of cells in a subject upon theadministration of an effective dosage of the modified compound.

Cytodegenerative diseases are characterized by the dysfunction and deathof cells, this dysfunction or death in the case of neurons leading tothe loss of neurologic functions mediated by the brain, spinal cord andthe peripheral nervous system. Examples of chronic neurodegenerativediseases include Alzheimer's disease, peripheral neuropathy (secondaryto diabetes or chemotherapy treatment), multiple sclerosis, amyotrophiclateral sclerosis, Huntington's disease and Parkinson's disease,Creutzfeldt-Jakob disease and AIDs dementia. Normal brain aging is alsoassociated with loss of normal neuronal function and may entail thedepletion of certain neurons. Examples of acute neurodegenerativedisease are stroke and multiple infarct dementia. Sudden loss of neuronsmay also characterize the brains of patients with epilepsy and thosethat suffer hypoglycemic insults and traumatic injury of the brain,peripheral nerves or spinal cord.

There continues to be a need for treatments that protect cells from celldeath resulting from episodes of, for example, disease, trauma,isolation and removal of tissues or cells from the body, or exposure totoxins. This need extends to, among other things: (i) treatments fornerve cell loss associated with chronic or acute neurodegenerativedisorders or trauma; (ii) treatments to minimize tissue damage resultingfrom ischemia where ischemia may occur as a result of stroke, heartdisease, a transplantation event, or other event resulting in a cut-offin nutritional supply to tissues; and, (iii) treatments to modulate celldeath associated with other degenerative conditions (such asosteoporosis or anemia). The absence of an effective cytoprotectivetherapy can result in either loss of life or a general decline in thequality of life, including permanent disability with high health carecosts to patients, their families and the health care providers.

There have been a number of experimental approaches and targetsevaluated to develop drugs for the protection of cells fromdegeneration. Glutamate, the main excitatory neurotransmitter in thecentral nervous system, is necessary for many normal neurologicalfunctions, including learning and memory. Overactivation of glutamatereceptors, however, results in excitotoxic neuronal injury, has beenimplicated in the pathogenesis of neuronal loss in the central nervoussystem (CNS) following several acute insults, includinghypoxia/ischemia. During brain ischemia caused by stroke or traumaticinjury, excessive amounts of the excitatory amino acid glutamate arereleased from damaged or oxygen deprived neurons. This excess glutamatebinds to the N-methyl-D-aspartate (NMDA) receptor which opens theligand-gated ion channel thereby allowing calcium influx, producing ahigh level of intracellular calcium which activates biochemical cascadesresulting in protein, DNA, and membrane degradation leading to celldeath. This phenomenon, known as excitotoxicity, is also thought to beresponsible for the neurological damage associated with other disordersranging from hypoglycemia and cardiac arrest to epilepsy. In addition,there are preliminary reports indicating similar involvement in thechronic neurodegeneration of Huntington's, Parkinson's and Alzheimer'sdiseases. Accordingly, many pharmaceutical strategies have been assessedwhich aim to decrease levels of glutamate excess.

Oxidative stress, caused by reactive oxygen species, represents anotherinjury mechanism implicated in many of the same acute and chronicdiseases. Reactive oxygen species (e.g., superoxide radical) would causeoxidative damage to cellular components, such as peroxidation of cellmembrane lipids, inactivation of transport proteins, and inhibition ofenergy production by mitochondria.

Glutamate excitotoxicity and oxidative stress may be interlinked;reactive oxygen species formation may occur as a direct consequence ofglutamate receptor overstimulation and thus mediate a component ofglutamate. Excitotoxicity, in turn, can be reduced by free radicalscavengers, including C, Zn-superoxide dismutase, the 21-aminosteroid“lazaroids”, the vitamin E analog, trolox, spin-trapping agents such asphenylbutyl-N-nitrone, and the ubiquinone analog, idebenone which reducethe amount of reactive oxygen species.

Mooradian has reported that certain estrogens have significantanti-oxidant properties in in vitro biochemical assays, but that thiseffect is not seen with all estrogens. (See, J. Steroid Biochem. Molec.Biol., 45 (1993) 509-511.) Because of the variation in anti-oxidantproperties noted by Mooradian in his biochemical assays, he concludedsteroid molecules must have certain anti-oxidant determinants which wereas yet unknown. Similar observations concerning steroids with phenolic Arings were reported in PCT Patent Application No. WO 95/13076, whereinbiochemical assays were used to show anti-oxidant activity. However, theassays used by Mooradian, as well as those used in WO 95/13076, werebiochemical assays and, as such, did not directly examine the effects ofthese molecules on cells. In contrast, Simpkins et al. describe, in U.S.Pat. No. 5,554,601 for example, cell-based assays to determine a methodof conferring neuroprotection on a population of cells using estrogencompounds based on demonstrated cell protective effects. As a result, inrecent years it has become recognized that estrogen, as well as otherpolycyclic phenols, may be used for this purpose. (See, e.g., U.S. Pat.Nos. 5,972,923; 5,877,169; 5,859,001; 5,843,934; 5,824,672; and,5,554,601; all of which are incorporated herein by reference.)

The mechanism by which estrogen compounds bring about a neuroprotectiveeffect is still not fully understood. However, these compounds have beenshown to have a number of different physiological and biochemicaleffects on neurons. For example, estrogen has been shown to stimulatethe production of neurotrophic agents that in turn stimulate neuronalgrowth. Estrogen compounds have also been found to inhibit NMDA-inducedcell death in primary neuronal cultures (see, e.g., Behl et al. Biochem.Biophys Res. Commun. (1995) 216:973; Goodman et al. J. Neurochem. (1996)66:1836), and further to be capable of removing oxygen free radicals andinhibiting lipid peroxidation (see, e.g., Droescher et al. WO 95/13076).For example, Droeschler et al. describe cell free in vitro assay systemsusing lipid peroxidation as an endpoint in which several estrogens, aswell as vitamin E, were shown to have activity. Estradiol has also beenreported to reduce lipid peroxidation of membranes (see, e.g., Niki(1987) Chem. Phys. Lipids 44:227; Nakano et al. Biochem. Biophys. Res.Comm. (1987) 142:919; Hall et al. J. Cer. Blood Flow Metab.(1991)11:292). Other compounds, including certain 21-amino steroids anda glucocorticosteroid, have been found to act as anti-oxidants and havebeen examined for their use in spinal cord injury, as well as headtrauma, ischemia and stroke. (See, e.g., Wilson et al. (1995) J. Trauma39:473; Levitt et al. (1994) J. Cardiovasc. Pharmacol 23:136; Akhter etal. (1994) Stroke 25; 418).

While anti-oxidant behavior is believed to be an important property, anumber of other factors are believed to be involved in achievingneuroprotection. As a result, it is to be noted that therapeutic agentsselected on the basis of a single biochemical mechanism may have limitedgeneralized utility in treating disease or trauma in patients. Forexample, in order to achieve an anti-oxidant effect in vitro usingestrogen, Droescher et al. used very high doses of estrogens. Suchdoses, even if effective on neurons in vivo, would have limited utilityin treating chronic neurological conditions because of associatedproblems of toxicity that result from the prolonged use of these highdosages.

In addition to the issues related to compound toxicity, considerationmust also be given to the ability of a particular compound to reach thetarget site, which in some applications is controlled by the ability ofthe compound to cross the blood-brain barrier. The blood-brain barrieris a complex of morphological and enzymatic components that retards thepassage of both large and small charged molecules, and thus limits theaccess of such molecules to cells of the brain. Furthermore, not onlymust the compound be capable of reaching the target site, but it mustalso do so in a state or configuration which enables it to carry-out itsdesignated function.

In view of the foregoing, it can be seen that a need continues to existfor the identification of compounds which have demonstrated biologicalefficacy in protecting humans from the consequences of abnormal celldeath in body tissue; compounds which are capable of crossing theblood-brain barrier and which are suitable for administration in dosageswhich are non-toxic. This identification requires continuing advances inthe understanding of the structural requirements for compositionscapable of inducing neuroprotection, which in turn provide the basis fordesigning novel drugs that have enhanced cytoprotective properties whileat the same time have reduced adverse side effects.

SUMMARY OF THE INVENTION

Among the several objects and features of the present invention includethe provision of a process for treating a population of cells againstcell death or cell damage wherein an effective dose of a cytoprotectiveor neuroprotective compound is administered thereto, the compound havinga hydroxy-substituted aromatic ring structure modified by, in someembodiments, (i) a non-fused polycyclic, hydrophobic substituent, or(ii) a bridged structure, a spiro structure (attached via a linker) or aring assembly; and, the provision of a process for treating acytodegenerative or neurodegenerative disease wherein an effective doseof such a compound is administered.

Further among the several objects and features of the present inventionis the provision of a compound having a hydroxy-substituted aromaticring structure modified as described above, and in some cases beingfurther modified by at least one other non-hydrogen substituent, thecompound having cytoprotective or neuroprotective activity; and, theprovision of such a compound wherein the hydroxy-substituted, aromaticring structure is also polycyclic.

Briefly, therefore, the present invention is directed to a process forconferring cytoprotection on a population of cells which comprisesadministering to that population of cells a compound comprising ahydroxy-substituted aromatic ring structure and a non-fused polycyclic,hydrophobic substituent attached thereto. The present invention isfurther directed to such a process wherein the cells are neurons.

The present invention is further directed to a process for treating acytodegenerative disease comprising administering to a subject (e.g.,human or animal) in need thereof a compound, or a pharmaceuticalcomposition comprising such a compound, comprising a hydroxy-substitutedaromatic ring structure and a non-fused polycyclic, hydrophobicsubstituent attached thereto.

The present invention is still further directed to one of the precedingprocesses wherein said compound has the formula:

wherein: n is 1 or 2; R¹ is a non-fused polycyclic, hydrophobicsubstituent; R^(x) is selected from the group consisting of hydrogen andsubstituted or unsubstituted alkyl; R¹³ is hydrogen or substituted orunsubstituted alkyl; and, R^(z) is hydrogen, hydroxy, substituted orunsubstituted alkyl, or oxo, with the proviso that when the compound hasthe following structure:

R^(x) is not hydrogen.

The present invention is still further to directed to a compound havingcytoprotective activity, the compound having the formula:

wherein: the compound optionally has one or more unsaturated bonds inconjugation with the aromatic A-ring between C-6 and C-7, C-8 and C-9 orC-9 and C-11, with the proviso that when C-8 or C-9 is unsaturated,R^(y) is not bound thereto; n ranges from 1 to 3; R¹ is a non-fusedpolycyclic, hydrophobic substituent; R^(x) is selected from the groupconsisting of hydrogen and substituted or unsubstituted alkyl; R^(y) andR^(v) are independently selected from hydrogen, substituted orunsubstituted alkyl, halo, amido, sulfate, and nitrate; p and q rangefrom 1 to 3; R¹³ is hydrogen or substituted or unsubstitutedhydrocarbyl, halo, amido, sulfate or nitrate; R^(z) is hydrogen,hydroxy, substituted or unsubstituted alkyl, or oxo; and, t ranges from1 to 3; with the proviso that when the compound has the followingstructure:

R^(x) is not hydrogen. The present invention is still further directedto one of the preceding processes wherein this compound is administeredin accordance therewith.

Other objects and features of the present invention will be in partapparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C generally illustrate chemical structures of somepreferred hydroxy-substituted aromatic compounds (e.g., phenols,catechols, etc., wherein n=1, 2 or more), which can be modified with alarge, hydrophobic substituent as described herein in accordance withthe present invention and which may be used to confer cytoprotection toa population of cells upon the administration of an effective dosethereof.

FIGS. 2A and 2B generally illustrate chemical structures of, in someembodiments, preferred polycyclic, hydrophobic substituents (e.g.,bridged substituents), as well as some alternative hydrophobicsubstituents (e.g., bridge-containing or ring assembly substituents)suitable for use in the present invention.

FIG. 3 generally illustrates chemical structures of some alternativehydroxy-substituted aromatic compounds (e.g., phenols, catechols, etc.,wherein n=1, 2 or more) wherein the modifying substituent R¹ is notattached directed to the hydroxy-substituted aromatic ring, but ratheris proximate to said ring, and further wherein said substituent R¹ isattached via a linker (e.g., alkenylene linker).

FIG. 4 generally illustrates certain embodiments of the compounds of thepresent invention, wherein other substituents (e.g., R², R³, R⁴, R⁵,etc.) attached to the terminal hydroxy-substituted, aromatic ring arefused to form polycyclic ring structures (i.e., structures having 2, 3or more rings, such as those illustrated in FIGS. 1A-1C).

FIG. 5 is a bar graph which, as further described in Example 3 below,illustrates the results of analyses performed to examine the impact acompound of the present invention had on cell death, at varying dosages.

FIG. 6 is a bar graph which, as further described in Example 3 below,illustrates the results of analyses performed to examine the impactvarying exposure times (to a compound of the present invention) had oncell death.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is now recognized that certain polycyclic phenolic compounds, inparticular estrogen-based compounds, have cytoprotective, and in somecases neuroprotective, activity (see, e.g., U.S. Pat. Nos. 5,972,923;5,877,169; 5,859,001; 5,843,934; 5,824,672; 5,554,601; 6,197,833; and,6,207,658; all of which are incorporated herein by reference). Withoutbeing held to a particular theory, it is generally believed that theactivity associated with estrogen compounds is, at least in part, aresult of the ability of estrogens, or more generally polycyclicphenolic compounds, because of their lipophilic nature, to becomeinserted into the cell membrane. Once in this position, the intactphenol group can donate a hydroxy hydrogen radical to prevent thecascade of membrane lipid peroxidation. Furthermore, it is generallybelieved that the significant potency of estrogens is because of theirability to donate a hydroxy hydrogen radical from several positions onthe A ring (see, e.g., U.S. Pat. No. 5,972,923), and because arelatively stable, oxidized estrogen is formed as a result of thishydrogen radical donation (due to the effects of resonance stability).

Surprisingly, it has now been discovered that these compounds, as wellas dihydroxy (e.g., catechol), trihydroxy, etc. analogs thereof, may bemodified by means of attaching a large or bulky hydrophobic substituenton the hydroxy-substituted ring, or alternatively at some other positionproximate to the hydroxy group, yielding compounds which are alsocapable of conferring cytoprotection to a population of cells (e.g.,neurons). In fact, Applicant's experimental data suggests the additionof such substituents, such as for example bridged polycyclicsubstituents, can act to enhance the cytoprotective activity of thesecompounds, relative to their respective non-substituted analogs.

Modified Phenolic or Catecholic Compounds

As stated above, contrary to expectations, it has been discovered thatpreviously reported phenolic compounds, as well as dihydroxy (e.g.,catecholic) analogs thereof, can be modified by means of attaching alarge, hydrophobic substituent (such as, for example, a polycyclicsubstituent comprising a bridged structure, a spiro structure attachedvia a linker, or a ring assembly), on or proximate to thehydroxy-bearing ring, in order to obtain a novel class of compoundshaving enhanced cytoprotective, and in some cases neuroprotective,activity. More specifically, it has been discovered that compoundshaving the formula (I):X—R¹  (I)wherein X generally represents the core or central structure, which inone exemplary embodiment is a phenol (such as those disclosed in, forexample, U.S. Pat. Nos. 5,972,923; 5,877,169; 5,859,001; 5,843,934;5,824,672; 5,554,601; 6,197,833; and, 6,207,658; all of which areincorporated herein by reference) and in a second exemplary embodimentis a catechol (such as the dihydroxy analogs of those moleculesdisclosed in the aforementioned U.S. patents), to which the modifyinghydrophobic substituent R¹ is attached, are suitable for use intreatments that protect a population of cells from cell death resultingfrom episodes of, for example, disease, trauma, isolation and removal oftissues or cells from the body, or exposure to toxins.

Generally speaking, the core structure, X, may represent essentially anycompound possessing a hydroxy-substituted aromatic ring. In a firstembodiment, X represents a compound having a terminal phenol ring while,in a second embodiment, X represents a compound having a terminal,dihydroxy-substituted (e.g. catechol) ring. More specifically, in theseembodiments, the present invention is directed to compounds having thegeneral formula (II)

wherein: n=1 or 2, for the first and second embodiments respectively; R¹is as previously noted and as further defined herein; and, R² and R³ arehydrogen or some other substituent, also as further described herein(including wherein these two are bound to different carbon atoms on thehydroxy-substituted ring, these substituents and the carbon atoms towhich they are bound forming a second, fused ring); X being generallyrepresented by formula (III)

In this regard it is to be noted that, as further described herein, inalternative embodiments X represents a polycyclic compound; that is, Xrepresents a compound having two or more (i.e., 2, 3, 4, 5 or more)hydrocarbon ring structures, or heterohydrocarbon ring structures(wherein a heteroatom is present in the ring), which may be fused orbound by some linkage, provided the terminal ring bears one or morehydroxy groups (e.g., phenol, catechol, etc.). In some particularlypreferred embodiments, as further described herein, X may be asteroid-like structure.

Additionally, it is to be noted that such polycyclic compounds mayoptionally have a polar or hydrophilic substituent that is at or nearthe end which is essentially opposite the hydrophobic substituent, R¹,thus rendering the overall compound (i.e., X—R¹) amphipathic. Such polaror hydrophilic substituents include, for example, oxo and hydroxy, aswell alkoxy or alkyloxy (wherein for example a hydroxy substituent hasbeen used to form an ether or has been esterified, by means common inthe art).

Referring now to FIGS. 1A, 1B and 1C, examples of core structure, X,suitable for use in the present invention include: (i) linked, two-ringstructures such as stibesterols (e.g., dimethylstibesterol,diethylstibesterol, dimethylstibesterol-mono-O-methyl, anddiethylstibesterol-mono-O-methyl); (ii) non-steroidal structures havinga terminal hydroxy-bearing ring and at least two additional hydrocarbonring structures, such as three-ring compounds (e.g.,[2S-(2a,4aα,10αβ)]-1,2,3,4,4a,9,10,10a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol(PAM) and[2S-(2a,4aα,10αβ)]-1,2,3,4,4a,9,10,10a-octahydro-7-hydroxy-2-methyl-2-phenanthrenecarboxyaldehyde(PACA)); (iii) compounds having a terminal hydroxy-bearing ring and atleast three additional carbon rings (e.g., 3,17α-estradiol;3,17β-estradiol; estratriene-3-ol; 2-hydroxy-17α-estradiol;2-hydroxy-17β-estradiol; estrone, 2-hydroxy estrone; estriol; 2-hydroxyestriol; ethynyl estradiol; and, 2-hydroxy ethynyl estradiol).

It is to be noted in this regard that the above-referenced listing ofcompounds is not intended to be exhaustive. For example, the position ofthe hydroxy group or groups on the terminal ring is not, in all cases,narrowly critical; that is, in some cases, the hydroxy group may occupyessentially any available position (which, depending upon the particularstructure of X, may be the 1, 2, 3, 4, etc. position on the terminalring). Additionally, in some cases, it may be favorable for X to containadditional double bonds in conjugation with the hydroxy-bearing aromaticring (such as in the case of distilbesterol compounds), for example asin the case of polycyclic compounds having the general structure (IV):

wherein a carbon-carbon double bond is present between C-6 and C-7, C-8and C-9, C-9 and C-11, or one of the possible combinations thereof.

Without being held to any particular theory, it is generally believedthis additional conjugation is favorable because it allows for theformation of a more stable, oxidized form of the compound; that is, itallows for additional delocalization of the phenoxy radical, which isbelieved to be formed as a result of the loss of a hydrogen radical toquench hydroperoxides (formed by the interaction of oxygen radicalspecies with unsaturated fatty acids). Accordingly, X may be other thanherein described without departing from the scope of the presentinvention.

In accordance with the present invention, it has been discovered thatthe core structure, X, may be modified by the addition of one or morelarge, hydrophobic substituents, R¹, in order to achieve enhancedcytoprotective activity, relative to the non-substituted analogs thereof(as further discussed and illustrated herein). More specifically, it hasbeen found that the cytoprotective activity of, for example, theabove-described phenolic compounds (such as those described by Simpkinset al.), as well as the dihydroxy-analogs thereof, can be enhanced bythe attachment of such a substituent either (i) on the terminal,hydroxy-bearing ring, or (ii) at some other position proximate thehydroxy group of the ring (see, e.g., FIG. 3). Furthermore, thehydrophobic substituent, R¹, can be attached directly or by some linkage(as further described herein; see also FIG. 3).

Generally speaking, it has been found that enhanced cytoprotectiveactivity is achieved when, in one embodiment, R¹ is a bridgedpolycyclic, hydrophobic substituent. Referring now to FIG. 2A, suitablesubstituents include, for example, bicyclic, tricyclic, tetracyclic,etc. structures comprising about 4, 6, 8, 10, 12 or more carbon atoms,such as: bicyclo[1.1.0]butanyl; bicyclo[2.2.1]heptanyl (i.e.,norbornyl); bicyclo[3.2.1]octanyl; bicyclo[4.3.2]nonanyl;bicyclo[4.3.2]undecanyl; tricyclo[2.2.1.0¹]heptanyl;tricyclo[5.3.1.1¹]dodecanyl; tricyclo[3.3.1.13,7]decanyl (i.e.,adamantyl); tricyclo[5.4.0.0^(2,9)]undecanyl; and,tricyclo[5.3.2.0^(4,9)]dodecanyl.

Without being held to a particular theory, this enhanced activity isbelieved to be, at least in part, a result of the hydrophobicsubstituent R¹ taking up a position within the void region of the cellmembrane or lipid bilayer, thus acting to “anchor” the compound andorient it such that the hydroxy-bearing aromatic ring (e.g., phenol orcatechol) is positioned proximate double bonds in the fatty acids of thelipid chains. The enhancement in the effectiveness or activity of thepresent compounds is therefore believed to be a result of the fact that,because of their composition and structure, these compounds arenaturally positioned within the environment to which they are deliveredat a location which optimizes their effectiveness.

Furthermore, it is generally believed that orientation of the presentcompounds within the lipid bilayer or cell membrane may be furtheraided, in some cases, by the addition or attachment of a polar orhydrophilic group at or near the end of the compound which issubstantially opposite the end to which the hydrophobic substituent R¹is attached. Other factors which may also impact orientation of thecompound include: (i) a substantially planar core structure X, andadditionally the entire compound, (which is believed to enhance theperformance of the present compounds); and/or (ii) the absence of apolar or hydrophilic substituent (R², R³, etc.) at a centrally locatedposition on the compound (which is believed to detrimentally impactperformance). For example, in the case of estrogen-like compounds,Applicant's experience to-date suggests changing the stereochemistry onthe B or C ring by, for example, opening the B ring decreases activity,and the presence of a polar group on the B or C ring reduces activity aswell.

Accordingly, essentially any large, hydrophobic substituent whichachieves the desired result (i.e., which acts to “anchor” the compoundand orient it at an optimal position within the lipid bilayer or cellmembrane) may be employed in accordance with the present invention. Morespecifically, it is generally believed that essentially any substituentmay be employed provided it is: (i) sufficiently hydrophobic in nature,such that it will be “drawn” or “pulled” into the hydrophobic portion ofthe bilayer or membrane; (ii) sufficiently large, such that once in thehydrophobic region it disturbs fatty acids in the membrane, thusdisrupting membrane integrity to a degree which results in thesubstituent being forced into the membrane void; and, (iii) sufficientlylong, either by itself or by the use of a linker, such that thehydroxy-substituted ring is positioned proximate the fatty acid doublebonds. Referring now to FIG. 2B, examples of suitable alternativeembodiments of R¹ include non-planar, polycyclic substituents, such asbridged structures or ring assemblies, which may be used alone or whichmay additionally be fused with another ring structure.

With respect to the position of the hydroxy-substituted ring, it is tobe noted that a typical membrane phospholipid has a length ranging fromabout 20 to about 30 Å (angstroms), as measured from about the end ofthe polar head group to about the end of the C-16 alkyl chain (by meansof molecular modeling programs standard in the art). Accordingly, it isto be noted that in such cases essentially any combination of (i) aterminal hydroxy-bearing aromatic ring structure (e.g., phenol orcatechol), and (ii) a hydrophobic substituent on or proximate to thehydroxy group on the aromatic ring structure, may be employed in thepresent invention, provided the distance between about the end of thehydrophobic “anchoring” substituent R¹ and about the opposite end of thehydroxy-bearing ring ranges from about 10 to less than about 30 Å (i.e.,about 15 Å, 20 Å, 25 Å).

Referring now to FIG. 3, it is to be further noted that, as previouslymentioned, while the “anchoring” substituent is typically attacheddirectly to hydroxy-bearing aromatic ring, it may also be attachedproximate this hydroxy group or groups; that is, the point of attachmentof this substituent is not narrowly critical in all applications,provided that in such applications the point of attachment of thesubstituent is sufficient to orient the compound in such a way that thedesired enhancement in activity is achieved. Accordingly, thesubstituent R¹ may be 1, 2, 3, 4 or more carbons from thehydroxy-bearing carbon, in some cases. Typically, however, thesubstituent R¹ will be adjacent or alpha to the hydroxy-bearing carbon.For example, in certain preferred embodiments, the hydroxy group is at a2 or 3 position, which means R¹ is preferably in a 2, 3 or 4 position.

It is to be still further noted that, as illustrated below (as well asin FIG. 3), the anchoring substituent R¹ may be directly attached to thecore molecule or it may alternatively be attached via a linker (“L”),provide the linker is of a length sufficient to generally position thehydroxy-bearing aromatic ring structure as described above.

Typically, a hydrocarbylene linker (e.g., alkyl, alkenyl, alkynyl) willbe employed having a length ranging from about 1 to about 6 carbons inthe main chain (e.g., methylene, ethylene, ethenylene, propylene,propenylene, etc.), with 1, 2 or 3 carbons in the main chain beingpreferred in some instances. Alternatively, however, in someembodiments, a hetero-substituted hydrocarbylene linker (such as anether or thioether) may be employed.

Additional and/or Alternative Substitution

In addition to the presence of one or more hydrophobic substituents, R¹,as described herein, it is to be noted that one or more othersubstituents (e.g., R², R³, R^(x), etc.), which may be the same ordifferent, may be attached to the hydroxy-bearing aromatic ring, oralternatively to some other segment or portion of the core structure X,provided the hydroxy-bearing ring remains in a substantially terminalposition in the overall compound (i.e., X—R¹) structure; that is, it isto be noted that the present compound may optionally contain one or moreadditional substituents, which may be the same or different, at variousavailable positions on the core structure, X, as illustrated belowwherein X is a polycyclic structure (e.g., an estrogen derivative)having the formula (V):

These other substituents (e.g., R^(y), R^(v), R^(z)) may, in someembodiments, be independently selected from the group consisting ofhydrogen, substituted or unsubstituted hydrocarbyl (e.g., methyl, ethyl,propyl, propenyl, butyl, etc.), halogens (e.g., fluoro, bromo, chloro),amides, sulfates, and nitrates (wherein p, q and t generally representthe number of such substituents present on, in this case, the B, C and Drings respectively, and which typically range from 0 to 2 for the B andC rings, and 0 to 3 for the D ring).

Furthermore, referring now to FIG. 4, as previously noted, thesubstituents (e.g., R² and R³) may, in combination with the carbon atomsto which they are bound, form a second, fused ring (e.g., a 5, 6, 7,etc. membered hydrocarbyl or heterohydrocarbyl) structure with theterminal, hydroxy-bearing ring. This second ring may, in turn, befurther substituted in the same way as described herein with referenceto the terminal hydroxy-bearing ring; that is, the second ring may haveone or more substituents (e.g., R⁴, R⁵, etc.) independently selectedfrom the group provided for R² and R³, which means X may in someembodiments comprise a third, forth, fifth, etc. ring structure (asshown, for example, in FIGS. 1A, 1B and 1C and as further describedherein).

In some preferred embodiments, substituents R², R³, R⁴, R⁵, etc., aswell as R^(y), R^(v), R^(z)) may be, for example:

(a) Alkyl, alkenyl, alkynyl, containing up to about six carbon atoms inthe main chain or ring structure (e.g., methyl, ethyl, propyl, butyl,pentyl, hexyl, dimethyl, isobutyl, isopentyl, tert-butyl, sec-butyl,methylpentyl, neopentyl, isohexyl, ethenyl, propenyl, butenyl, pentenyl,hexenyl, hexadienyl, 1,3-hexadiene-5-ynyl, isopropenyl, ethynyl,ethylidenyl, vinylidenyl, isopropylidenyl); sulfate; mercapto;methylthio; ethylthio; propylthio; methylsulfinyl; methylsulfonyl;thiohexanyl; thiopentyl; thiocyanato; sulfoethylamido; thionitrosyl;thiophosphoryl; p-toluenesulfonyl; amino; imino; cyano; carbamoyl;acetamido; hydroxyamino; nitroso; nitro; cyanato; selecyanato;arccosine; pyridinium; hydrazide; semicarbazone; carboxymethylamide;oxime; hydrazone; sulfurtrimethylammonium; semicarbazone;O-carboxymethyloxime; aldehyde hemiacetate; methylether; ethylether;propylether; butylether; benzylether; methylcarbonate; carboxylate;acetate; chloroacetate; trimethylacetate; cyclopentylpropionate;propionate; phenylpropionate; carboxylic acid methylether; formate;benzoate; butyrate; caprylate; cinnamate; decylate; heptylate;enanthate; glucosiduronate; succinate; hemisuccinate; palmitate;nonanoate; stearate; tosylate; valerate; valproate; decanoate;hexahydrobenzoate; laurate; myristate; phthalate; hydroxy;ethyleneketal; diethyleneketal; chloroformate; formyl; dichloroacetate;keto; difluoroacetate; ethoxycarbonyl; trichloroformate;hydroxymethylene; epoxy; peroxy; dimethyl ketal; acetonide; cyclohexyl;benzyl; phenyl; diphenyl; benzylidene; and, cyclopropyl. Thesubstituent(s) may in some embodiments be attached to any of theconstituent rings of X (i.e., the hydroxy-substituted ring or anotherring bound or fused thereto) to form, for example, a pyridine, pyrazine,pyrimidine, or v-triazine. The substituent(s) may also include, forexample, any of the six member or five member rings in section (b),below.

(b) A cyclic or heterocyclic carbon ring, which may be an aromatic ornon-aromatic ring and which may be bound (directly or via a linker) orbused with the hydroxy-substituted ring. This cyclic or heterocyclicring may optionally be substituted with any substituent described in (a)above. This additional ring structure, alone or in combination with thehydroxy-substituted A-ring, may be selected from, for example, one ormore of the following structures: phenanthrene; naphthalene; napthols;diphenyl; benzene; cyclohexane; 1,2-pyran; 1,4-pyran; 1,2-pyrone;1,4-pyrone; 1,2-dioxin; 1,3-dioxin (dihydro form); pyridine; pyridazine;pyrimidine; pyrazine; piperazine; s-triazine; as-triazine; v-triazine;1,2,4-oxazine; 1,3,2-oxazine; 1,3,6-oxazine(pentoxazole); 1,2,6-oxazine;1,4-oxazine; o-isoxazine; p-isoxazine; 1,2,5-oxathiazine;1,2,6-oxathiazine; 1,4,2-oxadiazine; 1,3,5,2-oxadiazine; andmorpholine(tetrahydro-p-isoxazine). Additionally, any of the abovecarbon ring structures may be linked directly or via a linkage group toa heterocyclic aromatic or nonaromatic carbon ring, such as: furan;thiophene(thiofuran); pyrrole(azole); isopyrrole(isoazole);3-isopyrrole(isoazole); pyrazole(1,2-daizole);2-isoimidazole(1,3-isodiazole); 1,2,3-triazole; 1,2,4-triazole;1,2-diothiole; 1,2,3-oxathiole, isoxazole(furo(a)monozole);oxazole(furo(b)monazole); thiazole; isothiazole; 1,2,3-oxadiazole;1,2,4-oxadiazole; 1,2,5-oxadiazole; 1,3,5-oxadiazole;1,2,3,4-oxatriazole; 1,2,3,5-oxatriazole; 1,2,3-dioxazole;1,2,4-dioxazole; 1,3,2-dioxazole; 1,3,4-dioxazole; 1,2,5-oxathiazole;1,3-oxathiole; and, cyclopentane. These compounds in turn may haveassociated substituent groups selected from section (a) or section (b)that are substituted on the ring at any of the available sites.

In this regard it is to be further noted that, in some embodiments,R^(z) may be a cycloalkyl or cycloalkenyl (e.g., cyclopentyl,cyclopentenyl), or alternatively alkoxyl (wherein an ether substituentis present on, for example, the D-ring of the structure, including forexample C1 to C8 alkoxy substituents, such as methoxy, ethoxy, propoxy,butoxy, pentoxy, etc.) or alkyloxy (wherein an ester substituent ispresent thereon). Additionally, R^(z) may be a spiro structure, whereina carbon of the ring to which it is attached is also a carbon of thecyclic structure. In a preferred embodiment t is 2 (i.e., 2 R^(z)substituents are present), R^(z) being a hydroxy or oxo substituent incombination with a spiro (e.g., cyclopentyl) substituent.

The core hydroxy-substituted structure, X, of the present invention maybe a cyclopentanophen(a)anthrene ring compound, for example selectedfrom the group consisting of the hydroxy-substituted analogs of:1,3,5(10),6,8-estrapentaene; 1,3,5(10),6,8,11-estrahexaene;1,3,5(10),6,8,15-estrahexaene; 1,3,5(10),6-estratetraene;1,3,5(10),7-estratetraene; 1,3,5(10),8-estratetraene;1,3,5(10),16-estratetraene; 1,3,5(10),15-estratetraene;1,3,5(10)-estratriene; and 1,3,5(10),9(11)-estratetraene.

The present invention is further directed to any compound as describedherein, as well as to the administration thereof to treat acytodegenerative disease, including precursors or derivatives selectedfrom raloxifen, tamoxifen, androgenic compounds, as well as their salts,where an intact hydroxy-bearing aromatic ring is present, with a hydroxygroup present on carbons 1, 2, 3 and 4 of the terminal phenol ring.

These compounds may be in the form of a prodrug, which may bemetabolized to form an active polycyclic phenolic, or catecholic,compound having cytoprotective, and in some cases neuroprotective,activity.

With respect to additional substituents (e.g., R², R³, R⁴, R⁵, R^(x),R^(y), R^(v), R^(z), etc.), it is to be noted that when such additionalsubstituents are present on the terminal, hydroxy-substituted aromaticring, or proximate thereto, in some embodiments it is preferred thatthese substituents be small (e.g., methyl, ethyl, propyl, butyl),relative to the size of the R¹ substituent of the present invention.Stated another way, it is preferred that in some embodiments of thepresent invention only one large, hydrophobic group be attached to thehydroxy-bearing aromatic ring, or proximate thereto.

Additionally, it is to be noted that when a smaller substituent ispresent, it is preferred that this substituent likewise be hydrophobicin nature. Again, without being held to a particular theory, it isgenerally believed that the presence of a hydrophilic group mayinterfere with the position and/or orientation of the overall compoundwithin the cell membrane or lipid bilayer.

Finally, it is to be noted that substituent R¹ may itself besubstituted. For example, when R¹ is a non-fused polycyclic substituent,such as adamantyl, the adamantyl ring may optionally be substituted toincrease the hydrophobicity (such as by attaching a halogen).

Additional Preferred Embodiments 1. Modification of theHydroxy-Substituted Aromatic Ring Structure

As previously noted, the core or terminal hydroxy-substituted aromaticring structure may optionally be modified with one or more substituentsin addition to the R¹ substituent (e.g., the non-fused polycyclic,hydrophobic substituent). In one preferred embodiment, thehydroxy-substituted aromatic ring is additionally modified with asubstituted or unsubstituted hydrocarbyl (e.g., methyl, ethyl, propyl,butyl, etc.) substituent. More preferably, when R¹ is a non-fusedpolycyclic, hydrophobic substituent, such as adamantyl, thehydroxy-substituted aromatic ring is additionally modified with an alkylsubstituent (such as methyl, ethyl, propyl, methylpropyl, etc.) or analkenyl substituent (such as ethylene, propylene, methylpropylene,etc.). Even more preferably, these substituents are alpha or adjacent tothe hydroxy group on the aromatic ring; that is, preferably the hydroxygroup is between the two substituents, the two substituents occupyingcarbon atoms which are directly bound to the carbon atom occupied by thehydroxy group.

Additionally, it is to be noted that Applicant's experience to-dateindicates that, in an alternative embodiment, a non-polycyclic,hydrophobic substituent (e.g., an alkyl or alkenyl substituent) may beemployed to increase cytoprotective activity, relative to thenon-substituted analog thereof. More specifically, experience to-dateindicates that, in some embodiments, the hydroxy-substituted aromaticring structure may be modified by the attachment of one or more groupsselected from, for example, methyl, ethyl, propyl (e.g., n-propyl,isopropyl), propenyl, methylpropyl, methylpropenyl, butyl (e.g.,isobutyl, t-butyl). In a preferred embodiment, a large or bulky alkylgroup is employed, such as a t-butyl or a methyl-propyl group (orlarger). In another preferred embodiment, the hydroxy-substituted ringmay be modified with two such substituents, such as t-butyl/methyl orethyl, propyl or propenyl/methyl or ethyl, etc.

2. Polycyclic, Hydroxy-Substituted Aromatic Ring Structure

As previously noted, the core hydroxy-substituted aromatic ringstructure may be part of a larger, polycyclic core structure (e.g.,bicyclic, tricyclic, tetracyclic, etc.). In one preferred embodiment,the hydroxy-substituted core structure has the formula (VI):

wherein n is typically 1 or 2, R¹ is a large or bulky hydrophobicsubstituent (e.g., a non-fused polycyclic), the hydroxy-substituted ringbeing the terminal or A-ring of the structure, and R^(x), R^(y), R^(v)and R^(z) are as defined herein. More preferably, however, the compoundof the present invention has the formula (VII):

wherein: n is as defined above, R¹ is a non-fused polycyclic,hydrophobic substituent (or alternatively a bulky hydrocarbyl group,such as t-butyl, methylpropyl, etc.); R^(x) is selected from the groupconsisting of hydrogen and substituted or unsubstituted hydrocarbyl(e.g., alkyl); R¹³ is hydrogen or substituted or unsubstitutedhydrocarbyl (e.g., alkyl); and, R^(z) is one or more substituentsselected from hydrogen, hydroxy, substituted or unsubstituted alkyl, oroxo (R^(y), R^(v) being hydrogen). Some of the preferred combinations ofsuch substituents include:

R¹ R^(x) R^(z) adamantyl methyl oxo adamantyl methylpropyl hydroxylt-butyl hydrogen hydroxyl adamantyl hydrogen hydroxyl methylpropylhydrogen hydroxyl adamantyl hydrogen oxo methylpropenyl hydrogenhydroxyl t-butyl hydrogen oxo methylpropyl hydrogen oxo t-butyl methyloxo hydrogen methyl oxo methylpropenyl hydrogen oxo hydrogenethylpropenyl oxowherein for example R¹, R^(x) and R^(z) occupy the C-2, C-4 and C-17positions on the ring, respectively (R^(z) being in the alpha or betaposition when is it hydroxy, for example).

In this regard it is to be noted that the present invention encompassesa number of compounds having one or more chiral centers therein.Generally speaking, therefore, it is to be understood that theconfiguration at one or more of these chiral centers may change withoutdeparting from the scope of the intended invention; that is, it is to beunderstood that the present invention extend to compounds specificallyor generally described herein, as well as all related diastereomers andenantiomers (e.g., (i) the naturally-occurring estrogen configurationwherein, when present, substituents at the C-8, C-9, C-13 and C-14position are beta, alpha, beta and alpha, respectively, or (ii) thenon-naturally-occurring estrogen configuration wherein, when present,these substituents have the alpha, beta, alpha, beta configuration).

Administration/Application

Generally speaking, the process of the present invention involves thetreatment of a population of cells in a subject (e.g., animal or human),in order to confer cytoprotection to that population, by theadministration of an effective dose of the above-described compound.Experience to-date suggests such protection can be achieved at lowplasma concentrations, concentrations which can be significantly lowerthan those needed for the non-substituted (i.e., non-R¹ substituted)analogs of the present compounds. More specifically, a cytoprotective oreven a neuroprotective effect can be achieved, in some cases, at plasmaconcentrations of less than about 10 μM, 1 μM, 500 nM, 100 nM, 10 nM, oreven 1 nM (i.e., from about 0.1 nM to about 1 nM).

Administration of any of the compounds of the invention may be achievedby means standard in the art, and may include the use of a singlecompound or a mixture of cytoprotective compounds, their enantiomers ordiastereomers, or pharmaceutically acceptable salts thereof. Therecommended route of administration of the compounds of the presentinvention includes oral, intramuscular, transdermal, buccal, nasal,intravenous and subcutaneous. Methods of administering the compounds ofthe invention may be by dose or by controlled release vehicles.

Additionally, it is to be noted that, similar to the approach describedby Simpkins et al. in U.S. Pat. No. 5,972,923 (incorporated herein byreference), a pharmaceutical preparation may also include, in additionto one or more compounds of the present invention, an additionalantioxidant. As noted by Simpkins et al., in reference to compoundssimilar to those of the present invention, synergistic effects may beachieved in certain circumstances when such a combination is employed.For example, Simpkins et al. reports that estratrienes exhibitapproximately a 1000-5000 fold enhancement in their cytoprotectiveeffect when administered with the antioxidant, glutathione.

The present compounds are suitable, for example, in treating subjectssuffering from trauma, chronic degenerative diseases or acute diseasesuch as induced by an ischemic attack. Specific examples includeAlzheimer's disease, Parkinson's disease, stroke, ischemia, heart attackor angioplasty, or brain or spinal cord trauma, hypoglycemia, anoxia,burns or surgeries that result in the loss of nutrient flow to thetissues. Other diseases that may be treatable with compounds of thecurrent invention include: heart disease, including myocardialinfarction, ophthalmologic diseases including macular degeneration, lensor retinal degeneration, formation of cataracts and glaucoma,alcoholism, alcohol withdrawal, drug-induced seizures vascularocclusion, epilepsy, cerebral vascular hemorrhage, hemorrhage;environmental excitotoxins, dementias of all type, drug-induced braindamage and other systemic or acute degenerative diseases characterizedby necrotic or apoptotic cell death. To-date, there are no known curesand few therapies that slow the progression of these diseases. However,the present invention provides compounds which can be used astherapeutics or as prophylactics to treat, prevent or delay the onset ofsymptoms.

Certain embodiments of the present invention may further be applied tothe procedure of tissue transplantation, prior, during or after removalor reperfusion of cells, tissues or organs or during storage of thecells, tissues or organs and is applicable to any of the cells in thebody.

Preparation

Generally speaking, the compounds of the present invention may beprepared by means standard in the art. Specific details for thepreparation of certain preferred compounds are provided herein in theExamples, below.

Activity

The activity of the compounds of the present invention may be determinedby means standard in the art (see, e.g., U.S. Pat. Nos. 5,972,923;5,877,169; 5,859,001; 5,843,934; 5,824,672; and, 5,554,601; all of whichare incorporated herein by reference). Alternative methods fordetermining activity are described in detail herein in the Examples,below.

Definitions

As used herein, the following phrases or terms shall have the notedmeanings; however, it is to be understood that these definitions areintended to supplement and illustrate, not preclude or replace, thedefinitions known to those of skill in the art.

“Hydroxy-substituted aromatic” or “hydyroxy-bearing aromatic” structureor ring, as well as variations thereof, refers to a terminal ring of acompound of the present invention which is both aromatic and substitutedwith one or more hydroxy groups. It is therefore to be understood thatsuch phrases are intended to refer to compounds wherein the entirestructure is aromatic (e.g., naphthalene, anthracene, andphenanthracene), as well as to compounds wherein only the terminal ringis aromatic (e.g., indan and 1,2,3,4-tetrahydronaphthlene).

“Cytoprotection” refers to the protection of cells against cell death orcell damage that would otherwise occur in the absence of a protectiveagent, where the cell death or cell damage might be caused by anymechanical damage, nutritional deprivation (including oxygendeprivation), trauma, disease processes, damage due to exposure tochemicals or temperature extremes, aging or other causes.

“Neuroprotection” is one form of cytoprotection and refers to theinhibition of the progressive deterioration of neurons that lead to celldeath.

“Enhanced” cytoprotective or neuroprotective activity refers to theincrease in activity of the compounds of the present invention (i.e.,compounds having a large, hydrophobic substituent, R¹, attached), ascompared to the non-substituted (i.e., non-R¹ substituted) analogsthereof.

“Non-fused, polycyclic” refers to polycyclic systems other than fusedsystems, and is intended to encompass bridged and spiro systems, as wellas ring assemblies. (See, e.g., Naming and Indexing of ChemicalSubstances for Chemical Abstracts, a reprint of Appendix IV from theChemical Abstracts 1997 Index Guide, ¶¶ 147-155, pp. 260I-266I.)

“Fused systems” are polycyclic structures containing at least two ringsof five or more members having (i) only “ortho” fusion, whereinadjoining rings have only two atoms in common and thus have n commonfaces and 2n common atoms, such as naphthalene, or (ii) “ortho” and“peri” fusion, wherein a ring has two, and only two, atoms in commonwith each of two or more rings, the total system containing n commonfaces and fewer than 2n common atoms, such as pyrene. (See, e.g., Id.)

“Bridged systems” are monocyclic or fused systems with valence bonds,atoms or chains connecting different parts of the structure.

“Spiro systems” have pairs of rings (or ring systems) with only onecommon atom. (See, e.g., Id.)

“Ring assemblies” have pairs of rings (or ring systems) connected bysingle bonds. (See, e.g., Id.)

An “estrogen compound” refers to any of the structures described in the11th Edition of “Steroids” from Steraloids Inc., Wilton N.H.,incorporated herein by reference. Included in this definition areisomers and enantiomers, including non-steroidal estrogens formed bymodification or substitution of the compounds in the Steraloidreference. Other estrogen compounds included in this definition areestrogen derivatives, estrogen metabolites and estrogen precursors, aswell as those molecules capable of binding cell-associated estrogenreceptors as well as other molecules where the result of bindingspecifically triggers a characterized estrogen effect. Also included aremixtures of more than one estrogen, where examples of such mixtures areprovided in, for example, U.S. Pat. No. 5,972,923. Examples ofα-estrogen structures having utility either alone or in combination withother agents are provided in, for example, U.S. Pat. No. 5,972,923 aswell.

A “non-estrogen compound” refers to a compound other than an estrogencompound as defined above.

The terms “17-E2,” “β-estradiol,” “17β-stradiol,” “β-17-E2,” “17β-E2,”“E2,” “17βE2,” and “βE2,” are intended to be synonymous. Similarly, theterms “α17-E2,” “α-17-E2,” “α-estradiol,” “17α-estradiol,” “17αE2,” and“αE2,” as defined here and in the claims, are intended to be synonymousand correspond to the α-isomer of 17β-estradiol.

“E-3-ol” refers to estra-1,3,5(10)-trien-3-ol.

The terms “polycyclic phenolic compound,” “polycyclic compounds” or“polycyclic phenols” as used herein are generally synonymous and aredefined, for example, in U.S. Pat. No. 5,859,001 (herein incorporated byreference); the terms generally include any compound having a phenolic Aring and may contain 2, 3, 4 or even more additional ring structuresexemplified by the compounds described herein.

A “steroid” refers to a compound having numbered carbon atoms arrangedin a 4-ring structure (see, e.g., J. American Chemical Society,82:5525-5581 (1960); and, Pure and Applied Chemistry, 31:285-322(1972)).

A “cytodegenerative” disorder or disease refers to a disorder or diseaserelated to cell death or cell damage, which might be caused by anymechanical damage, nutritional deprivation (including oxygendeprivation), trauma, disease processes, damage due to exposure tochemicals or temperature extremes, aging or other causes.

A “neurodegenerative disorder” or “neurodegenerative disease” refers toa disorder or disease in which progressive loss of neurons occurs eitherin the peripheral nervous system or in the central nervous system.Examples of neurodegenerative disorders include: chronicneurodegenerative diseases, such as Alzheimer's disease, Parkinson'sdisease, Huntington's chorea, diabetic peripheral neuropathy, multiplesclerosis, amyotrophic lateral sclerosis; aging; and acuteneurodegenerative disorders including: stroke, traumatic brain injury,schizophrenia, peripheral nerve damage, hypoglycemia, spinal cordinjury, epilepsy, and anoxia and hypoxia.

“Linker” embraces a saturated or partially unsaturated moiety, typicallya hydrocarbylene (e.g., alkylene, akenylene, akynylene), oralternatively a hetero-substituted hydrocarbylene (e.g., wherein acarbon in the main chain has been substituted by a heteroatom, such asoxygen or sulfur), interposed between the core ring structure X and themodifying hydrophobic substituent, R¹, or alternatively between the corering structure X and another substituent (e.g., R², R³, etc.).

“Hydrocarbyl” embrace moieties consisting exclusively of the elementscarbon and hydrogen, in a straight or branched chain, or alternatively acyclic structure, which may optionally be substituted with otherhydrocarbon, halo (e.g., chlorine, fluorine, bromine) or hetero (e.g.,oxygen, sulfur) substituents. These moieties include alkyl, alkenyl,alkynyl and aryl moieties, as well as alkyl, alkenyl, alkynyl and arylmoieties substituted with other aliphatic or cyclic hydrocarbon groupssuch as, for example, alkaryl, alkenaryl and alkynaryl.

The alkyl groups described herein are, in some embodiments, preferablylower alkyl containing from about 1 to about 6 carbon atoms in theprincipal chain. They may be straight or branched chains and includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,pentyl, hexyl and the like. They may be substituted with aliphatic orcyclic hydrocarbon moieties or hetero-substituted with the varioussubstituents defined herein.

The alkenyl groups described herein are, in some embodiments, preferablylower alkenyl containing from about 2 to about 6 carbon atoms in theprincipal chain. They may be straight or branched chains and includeethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl,and the like. They may be substituted with aliphatic or cyclichydrocarbon moieties or hetero-substituted with the various substituentsdefined herein.

The alkynyl groups described herein are, in some embodiments, preferablylower alkynyl containing from about 2 to about 6 carbon atoms in theprincipal chain. They may be straight or branched chain and includeethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like.They may be substituted with aliphatic or cyclic hydrocarbon moieties orhetero-substituted with the various substituents defined herein.

The term “cycloalkyl” is used herein to refer to a saturated cyclicnon-aromatic hydrocarbon moiety having a single ring or multiplecondensed rings. Exemplary cycloalkyl moieties include, for example,cyclopentyl, cyclohexyl, cyclooctanyl, etc.

The term “cycloalkenyl” is used herein to refer to a partiallyunsaturated (i.e., having at least one carbon-carbon double bond),cyclic non-aromatic hydrocarbon moiety having a single ring or multiplecondensed rings. Exemplary cycloalkenyl moieties include, for example,cyclopentenyl, cyclohexenyl, cyclooctenyl, etc.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refer tocycloalkyl and cycloalkenyl moieties, respectively, as just describedwherein one or more hydrogen atoms to any carbon of these moieties isreplaced by another group such as a halogen, alkyl, alkenyl, alkynyl,substituted alkyl, substituted alkenyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, cycloalkenyl, substituted cycloalkyl,substituted cycloalkenyl, heterocyclo, substituted heterocyclo,heteroaryl, substituted heteroaryl, alkoxy, aryloxy, boryl, phosphino,amino, silyl, thio, seleno and combinations thereof.

“Hydrophobic,” as used in the context of the substituent attached to thehydroxy-substituted aromatic ring structure (i.e., R1), generally refersto this substituent's affinity for nonaqueous environments, and morespecifically to its affinity for the hydrophobic region of a lipidbilayer upon introduction thereto.

“Terminal,” as used in the context of the hydroxy-substituted aromaticring structure, generally refers to the position of the ring relative tothe rest of the molecule, the ring being located at or proximate one endof the molecule, such as in the case of the tetracyclic estrogencompounds (the hydroxy-substituted aromatic ring being the A ring of thecompound).

The following Examples set forth one approach for preparing and testingcompounds in accordance with the present invention. These Examples areintended to be illustrative of compounds preferred for certainembodiments only, as well as their respective activity in the protectionof neuron. Generally speaking, however, it is understood that in manycases drugs which protect neurons are also active in protectingnon-neuronal cells. Accordingly, in advancing the understanding of thestructural requirements for compositions capable of inducingneuroprotection, these results in turn provide the basis for designingnovel drugs that have enhanced cytoprotective properties, as well.Therefore, these Examples should not be viewed in a limiting sense.

Example 1 Preparation of2-(1-Adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one

Estrone [3-hydroxyestra-1,3,5(10)-trien-17-one, 270 mg, 1 mmol) and1-adamantanol (170 mg, 1 mmol) were added to anhydrous n-pentane (6 mL)and the stirred mixture was cooled with an ice bath. Boron trifluorideetherate (BF₃.EtOEt, 0.4 mL) was added over a 10 minute period. After anadditional 15 minutes, the ice bath was removed and stirring wascontinued for an additional 45 minutes at room temperature. During the45 minute period, solids present in the reaction mixture were dissolvedand a yellow oil formed. Crushed ice was then added while shaking andswirling the reaction flask and a pink solid was formed. The filtered,crude pink product was washed with water until the filtrate had aneutral pH, and then the solid was dried in a vacuum oven at 50° C. Thecrude pink powder (0.4 g) was purified by flash chromatography (silicagel eluted with 20% ethyl acetate in hexanes) to get the pure product(0.31 g, 76.7%). The product was recrystallized from a mixture ofchloroform and isopropyl alcohol and characterized as follows: (i)melting point=322-324° C. (see, e.g., Lunn, W. H. W.; Farkas, E.,Adamantyl Carbonium Ion as a Dehydrogenating Agent: Its Reactions withEstrone, Tetrahedron 1969, 24, 6773-76, citing melting point as 295-296°C.); (ii) ¹H NMR (CDCl₃, 300 MHZ) δ0.94 (s, 3H, C₁₈—CH₃), 2.8 (m, 2H,C₆CH₂), 4.7 (s, 1H, C₃—OH), 6.4 (5, 1H, C₄—H), 7.12 (5, 1H, C₁—H); (iii)¹³C NMR CDCl₃, 300 MHZ) δ3.76, 21.47, 25.93, 26.41, 28.63, 28.95 (3×C),31.56, 35.81, 36.56, 36.98 (3×C), 38.42, 40.69 (3×C), 44.25, 47.99,50.35, 116.87, 124.11, 131.59, 134.00, 135.02, 152.44, 221.43. (ChemicalAbstracts Registry Number [21003-01-0].)

Example 2 Preparation of(17β)-2-(1-Adamantyl)-estra-1,3,5(10)-triene-3,17-diol

2-(1-Adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (250 mg, 0.62mmol) was added to cold ethanol (25 mL) and methanol (10 mL) to give aturbid solution. Sodium borohydride (NaBH₄, 140 mg) was added in oneportion and the reaction was continued with stirring for 2 hours.Solvents were removed on a rotary evaporator and crushed ice was added.On standing overnight, the initially formed oil became a solid. Thesolid was filtered and washed with water until the filtrate was pHneutral. The solid was dried in a vacuum oven at 50° C. to give thecrude product (0.25 g), which was purified by flash chromatography(silica gel eluted with 18% ethyl acetate in hexanes). The pure product(200 mg, 79.6%) was recrystallized from chloroform and hexanes to obtaincrystals (150 mg), and was then characterized as follows: (i) meltingpoint=174-175° C.; (ii) ¹H NMR (CDCl₃, 300 MHZ) δ0.81 (s, 3H, C₁₈—CH₃),2.76 (m, 2H, C₆—CH₂), 3.73 (t, 1H, C₁₇—H), 4.78 (s, 1H, C₃—OH), 6.38 (s,1H, C₄—H), 7.16 (s, 1H, C₁—H); (iii) ¹³C NMR (CDCl₃, 300 MHZ) 60.95,23.02, 26.33, 27.12, 28.77, 28.98 (3×C), 30.49, 36.54, 36.71, 37.01(3×C), 38.89, 40.68 (3×C), 43.20, 44.22, 49.96, 81.99, 116.84, 124.06,132.06, 133.83, 135.20, 152.36.

Example 3 Comparative Study of the Activity of 17-β-Estradiol andAdamantyl-Modified Analog Thereof Method

Glial cell cultures: Glial cell cultures were prepared from P1 mice.Briefly, cortex from P1 mice were dissected and digested with 0.025%trypsin for 30-40 min. After trituration, the cells were plated onpoly-D-lysine/laminin coated glass-bottom dishes at a density ofapproximately 5×10⁶ cells per dish. Cells were grown for 7-10 days inEagle's minimal essential medium supplemented with 10% fetal bovineserum, 10% horse serum, 10 ng/ml epidermal growth factor, 2 mM glutamineand 20 mM glucose. Cultures were kept at 37° C. in a humidified CO₂atmosphere until they reached confluence. Cultures were then used tosupport neurons (see below).

Cortical neuronal cultures: Cortical neurons were prepared from E15-16mouse embryos. Dissociated cortical cells (at a density of 5×10⁶ cellsper dish) were plated on a layer of confluent glial cells (7-10 days invitro), into Eagle's minimal essential medium supplemented with 5% fetalbovine serum, 5% horse serum, 2 mM glutamine, and 21 mM glucose.Cultures were kept at 37° C. in a humidified 5% CO₂ atmosphere. After3-5 days in vitro, non-neuronal cell division was halted by exposure to10⁻⁵ M cytosine arabinoside for 2 days, and cultures were shifted to agrowth medium identical to the plating medium but lacking 10% fetalbovine serum. Cultures 9-14 days in vitro were used for the experiments.

Neuronal cell death: Mixed neuron/glial cultures were exposure to NMDA(30 μM) for 24 hours in medium stock (MS) (minimal essential medium with15.8 mM NaHCO₃ and 20 mM glucose, pH 7.4) at 37° C. Cell death wasassessed qualitatively by counting cells that stained with Trypan blue(0.4% for 10 minutes at 37° C.). Stained neurons were counted from threerandom fields per dish. For the experiments to test the neuroprotectiveeffect of the 17-β-estradiol and the adamantyl-modified analog thereof,cultures were incubated with the drugs for various time periods prior towhen NMDA was added to the medium. Results

As seen by others, 24 hour pre-incubation with 17-β-estradiol attenuatedcortical cell death induced by 24 hour exposure to 30 μM NMDA. We foundthat adamantyl-modified analog was more effective in protecting corticalneurons from excitotoxicity. As shown in FIG. 5, 10 μM 17-β-estradiolreduced neuronal loss due to NMDA exposure by about 15%, whereas 1 μM ofthe adamantyl-modified analog (denoted zyc-5) protected neuronal deathby about half; that is, at one-tenth the dosage, the adamantyl-modifiedcompound was found to be more than three times more effective. Theprotective effect of adamantyl-modified compound was dose-dependent,dropping to 10% with a 100 nM dosage.

Referring now to FIG. 6, cultured cortical neurons were pre-incubatedwith 1 μM of the adamantyl-modified compound for 1, 3, 6, 12 and 24hour(s) prior to NMDA exposure. There was no significant difference ofneuronal survival from 1-12 hours pre-incubation; 24 hourspre-incubation gave the best protection results.

Example 4 Preparation of3-Hydroxy-2-(1methylpropyl)-estra-1,3,5(10)-trien-17-one

To a solution of the known3-hydroxy-2-(1-methyl-2-propenyl)estra-1,3,5(10)-trien-17-one (ref.Patton, 1962; Chemical Abstracts Registry Number [98543-85-2], 30 mg,0.093 mmol) dissolved in 10 mL of anhydrous ethanol was added 20 mg of5% Pd/C. The reaction flask was shaken under 2.7 atm. of hydrogen for 3h. The reaction mixture was then filtered and the catalyst was washedwith ethanol. After solvent removal, the crude product was purified bychromatography (silica gel eluted with 12.5% ethyl acetate in hexanes)and then recrystallized from methylene chloride-hexanes to give 20 mg ofpure product: m.p. 186-187° C.; ¹H NMR(CDCl₃) δ0.85-0.90 (m, 3H, CH₃),0.91 (s, 3 h CH₃), 1.20-1.24 (m, 3H, CH₃), 2.81-2.89 (m, 2H, CH₂), 6.51(s, 1H, Ar—H), 7.06 (s, 1H, Ar—H); ¹³C NMR(CDCl₃) δ12.17, 13.75, 20.44,21.46, 25.93, 28.91, 29.83, 31.49, 33.87, 35.81, 38.33, 44.05, 48.01,50.24, 115.37, 123.99, 124.17, 130.86, 131.88, 134.78, 157.267, 221.79.

Example 5 Preparation of2-(1,1-Dimethylethyl)-3-hydroxy-4-methylestra-1,3,5(10)-trien-17-one

To a suspension of the known3-hydroxy-4-methylestra-1,3,5(10)-trien-17-one (ref. Kaneko et al.,1964; Chemical Abstracts Registry Number [68969-90-4], 30 mg, 0.11 mmol)in 1 mL of anhydrous pentane and 0.5 mL of t-butanol, was added 0.03 mLof boron trifluoride diethyl etherate while cooling with an ice bath andstirring. After 20 min., the reaction was stirred at room temperaturefor 2.5 h. Ice was added and the solid that formed was filtered, washedwith water and dried overnight in a vacuum desiccator. The crude productwas purified by chromatography (silica gel eluted with 10% ethyl acetatein hexanes) and then recrystallized from methylene chloride-hexanes togive 20 mg of pure product (56% yield): m.p. 190-192° C.; ¹H NMR(CDCl₃)δ0.90 (s, 3H, CH₃), 1.42 (s, 9H, C(CH₃)₃), 2.13 (s, 3H, CH₃), 2.79-2.81(m, 2H, CH₂), 7.16 (s, 1H, Ar—H); ¹³C NMR (CDCl₃) δ 11.06, 13.70, 21.44,26.16, 26.60, 26.68, 27.54, 29.82 (3×C), 31.55, 34.43, 35.84, 37.63,44.52, 47.89, 50.34, 121.42, 131.28, 133.00, 133.55, 150.56, 221.45.(Anal. calc'd. for C₂₃H₃₂O₂: C, 81.13; H, 81.09. Found: C, 81.09; H,9.59.)

Example 6 Preparation of2-(1-admantanyl)-3-hydroxy-4-methylestra-1,3,5(10)-trien-17-one

To a suspension of the known3-hydroxy-4-methylestra-1,3,5(10)-trien-17-one (ref. Kaneko et al.,1964; Chemical Abstracts Registry Number [68969-90-4], 30 mg, 0.11 mmol)and 30 mg (0.197 mmol) of 1-adamantanol in 1 mL of anhydrous pentane wasadded 0.02 mL of boron trifluoride diethyl etherate while cooling withan ice bath and stirring. After stirring at 0° C. for 15 min. thereaction was stirred at room temperature for 45 min. Ice was added andthe white solid that formed was filtered, washed with water and driedover P₂O₅. The crude product was purified by chromatography (silica geleluted with 7% ethyl acetate in hexanes) to give 30 mg of pure product(68% yield). After recrystallization from methylene chloride-hexanes theproduct had: m.p. 262-263° C.; ¹H NMR(CDCl₃) δ0.90 (s, 3H, CH₃), 2.13(s, 3H, CH₃), 2.7 (m, 2H, CH₂), 7.11 (s, 1H, Ar—H); ¹³C NMR (CDCl₃)δ11.02, 13.70, 21.44, 26.22, 26.67, 27.54, 28.95 (3∴C), 31.56, 35.84,36.60, 36.98 (3×C), 37.63, 40.82 (3×C), 44.60, 47.91, 50.34, 121.41,121.51, 131.45, 133.32, 150.79, 221.49. (Anal. calc'd. for C₂₉H₃₉O₂: C,83.21; H, 9.15. Found: C, 83.45; H, 8.94.)

Example 7 Preparation of(17β)-2-(1-Adamantyl)-4-(1-methylpropyl)estra-1,3,5(10)-triene-17-diol

(A two step synthesis was involved, the first compound being anintermediate and the second being the above-referenced compound): To amixture of the known3-hydroxy-4-(1-methylpropyl)estra-1,3,5(10)-trien-17-one (ref. Miller etal., 1996; Chemical Abstracts Registry Number [177353-06-9], 70 mg,0.215 mmol) and 50 mg (0.328 mmol) of 1-adamantanol in 2 mL of anhydrouspentane was added 0.1 mL of boron trifluoride diethyl etherate whilecooling with an ice bath and stirring. After 1 h, stirring was continuedat room temperature for an additional 1 h and a yellowish orangesuspension formed. After adding ice, the reaction mixture was extractedwith ethyl acetate. The combined extracts were washed with brine anddried over anhydrous sodium sulfate. After solvent removal, the crudeproduct was purified by chromatography (silica gel eluted with 6% ethylacetate in hexanes) to give 40 mg of product (40.5% yield) as a oil. ¹HNMR(CDCl₃) δ0.84-0.99 (m, 3H, CH₃), 0.90 (s, 3H, CH₃), 2.86-3.16 (m, 2H,CH₂); ¹³C NMR(CDCl₃) δ13.05, 13.35, 13.67, 13.72, 13.97, 18.30, 21.43,22.52, 26.22, 27.04, 27.45, 27.57, 28.29, 28.97 (3×C), 31.47, 31.62,33.82, 34.23, 35.84, 36.68, 36.98 (3×C), 37.33, 37.40, 40.96 (3×C),44.80, 47.85, 50.44, 122.01, 130.33, 132.92, 133.80, 152.49, 221.32.

To prepare the above-referenced compound, to a solution of 40 mg (0.087mmol) of2-(1-adamantyl)-3-hydroxy-4-(1-methylpropyl)estra-1,3,5(10)-trien-17-onein 5.5 mL of anhydrous methanol, at −10° C. was added 50 mg of sodiumborohydride in one portion and the stirring was continued at −5° C. for1 h. After solvent removal, ice was added and the solid that formed wasfiltered, washed with water and dried over P₂O₅. Purification bychromatography (silica gel eluted with 12.5% ethyl acetate in hexanes)gave 20 mg of purified product (50% yield) that had: m.p. 152-154° C.;1H NMR(CDCl₃) δ0.78 (s, 3H, CH₃), 0.85-0.95 (overlapping t, J=7.4 Hz,3H, CH₃), 1.34-1.36 (d, J=7.4 Hz, 3H, CH₃), 2.81-2.84 (m, 2H, CH₂), 3.74(t, J=8.1 Hz, 1H, CHOH), 7.12 (m, 1H, Ar—H); ¹³C NMR(CDCl₃) δ10.86,10.90, 13.06, 13.37, 18.30, 22.97, 26.60, 27.69, 28.32, 29.00 (3×C),30.58, 33.75, 34.17, 36.68, 36.78, 37.00 (3×C), 37.79, 37.86, 40.96(3×C), 43.07, 44.78, 44.82, 50.08, 81.93, 121.98, 130.27, 131.89,133.14, 133.67, 152.33.

Example 8 Preparation ofEnt-(17β)-2-(1-Adamantyl)estra-1,3,5(10)-triene-3,17-diol

A suspension of the known ent-17β-estradiol (ref. Green et al., 2001,Chemical Abstracts Registry Number [3736-22-9], 40 mg, 0.147 mmol) and20 mg of 1-adamantanol (0.13 mmol) in 1 mL anhydrous pentane was stirredat room temperature for 20 min. and then at −5° C. for 15 min. To thissuspension was added 0.05 mL of boron trifluoride diethyl etherate over1 min. Then it was stirred at 0° C. to −5° C. for 20 min. and a paleyellow solution was obtained. The reaction then was stirred at roomtemperature for 15 min., during which time a sticky substance wasformed. After 45 min., ice was added with stirring until the stickysubstance solidified. The solid was then filtered, washed with water anddried in a vacuum desiccator to yield 50 mg of crude product.Purification by chromatography (silica gel eluted with 20% ethyl acetatein hexanes) gave 40 mg of pure product (67% yield). Aftercrystallization from methylene chloride the pure product had: m.p.174-176° C.; [α](24, D) −198 (c=0.1, CHCl₃); ¹H NMR(CDCl₃) δ0.78 (s, 3H,CH₃); 2.75-2.76 (m, 2H, CH₂); 3.78 (t, J=8 Hz, 1H, CHOH); 6.39 (s, 1H,Ar—H); 7.15 (s, 1H, Ar—H); ¹³C NMR(CDCl₃) δ152.16, 135.16, 133.73,132.13, 124.02, 116.81, 81.98, 50.08, 44.31, 43.29, 40.82 (3×C), 39.01,37.10 (3×C), 36.81, 36.64, 30.65, 29.10 (3×C), 28.87, 27.22, 26.44,23.14, 11.07. (Anal. calc'd. for C₂₈H₃₈O₂: MS m/z 406(M⁺).)

Example 9 Preparation ofEnt-(17β)-2-(1,1-dimethylethyl)estra-1,3,5(10)-triene-3,17-diol

A suspension of the known ent-17β-estradiol (ref. Green et al., 2001,Chemical Abstracts Registry Number [3736-22-9], 30 mg, 0.11 mmol) and0.06 mL of 2-methyl-2-propanol (0.63 mmol) in 1 mL anhydrous pentane wasstirred at room temperature 15 min. and then at 0° C. to −5° C. for 20min. To this suspension was added 0.07 mL boron trifluoride diethyletherate over 1 min. and stirring was continued at 0° C. to −5° C. for20 min. The reaction was allowed to warm to room temperature and duringthis period (˜15 min.) a yellow solid that stuck to the flask wasformed. After stirring for 30 min. at room temperature, ice was added.The powder-like solid was filtered, washed with water and dried in avacuum desiccator over night. The crude product was purified bychromatography (silica gel eluted with 18% ethyl acetate in hexanes) toget the pure product which was then crystallized from acetone-hexane.The pure product (20 mg, 55% yield) had: m.p. 177-179° C.; [α](25, D)−91.33 (c=0.225, CHCl₃); ¹H NMR (CDCl₃) δ0.78 (5, 3H, CH₃), 1.40 (5, 9H,C(CH₃)₃), 2.74-2.75 (m, 2H, CH₂), 3.74 (t, J=8.4 Hz, CHOH), 6.41 (5, 1H,Ar—H); 7.19 (5, 1H, Ar—H); ¹³C NMR (CDCl3) δ52.05, 135.33, 133.37,131.81, 124.04, 116.55, 81.98, 60.44, 50.03, 44.23, 43.26, 38.98, 36.78,34.47, 30.57, 29.74 (3×C), 28.90, 27.22, 26.38, 23.12, 14.17, 11.07.(Anal. calc'd. for C₂₂H₃₂O₂: MS m/z 328(M⁺), 313.)

Example 10 Comparative Study of Compound Activity Method: HT-22 CellNeuroprotection Assay

HT-22 cells (immortalized hippocampal neurons of murine origin) weremaintained in DMEM media (Life Technologies, Inc., Gaitherburg, Md.)supplemented with 10% charcoal-stripped FBS (HyClone Laboratories, Inc.,Logan, Utah) and 20 μg/mL gentamycin, according to standard cultureconditions.

Cells were plated at a density of 5,000 cells/well in clear-bottomedNunc 96-well plates (Fisher Scientific, Orlando, Fla.) and allowed toincubate overnight. Steroids dissolved in DMSO were added atconcentrations ranging from 0.01-10 μM and were co-administered withglutamate (10 mM or 20 mM). DMSO was used at concentrations of 0.1%vol/vol as a vehicle control and had no discernible effect on cellviability. After ˜16 h of glutamate exposure, cells were rinsed withPBS, pH 7.4, and viability was assessed by the addition of 25 μM calceinAM (Molecular Probes, Inc., Eugene, Oreg.) in PBS for 15 min at roomtemperature. Fluorescence was determined (excitation 485, emission 530)using a fluorescence FL600 microplate reader (Biotek, Winooski, Vt.).Cells that were lysed by addition of methanol were used for blankreadings. All data were normalized to % cell death, as calculated by(control value−insult value)/control value×100.

Results

Test results are presented in Tables 1 and 2, below.

TABLE 1

Steroid concentration Steroid concentration needed to protect needed toprotect 50% 50% of neurons of neurons killed by killed by 20 mM 10 mMGlutamate ED₅₀ Glutamate D₅₀ Compound R₁ R_(x) R_(z) (μM) (μM)  1 ZYC-26Adamantyl Me ═O 0.018 0.030  2 ZYC-22 Adamantyl sec- β-OH 0.021 0.022Butyl  3 ZYC-15 t-Butyl H β-OH 0.045 0.14  4 ZYC-21 Sec-Butyl H β-OH0.16 0.29  5 ZYC-3 Adamantyl H ═O 0.17 0.38  6 ZYC-20 sec-But-2- H β-OH0.24 0.31 enyl  7 ZYC-14 t-Butyl H ═O 0.25 0.30  8 ZYC-19 Sec-Butyl H ═O0.29 0.32  9 ZYC-25 t-Butyl Me ═O 0.32 0.32 10 ZYC-5 Adamantyl H β-OH0.39 0.16 11 ZYC-17 H sec- ═O Not Done 0.33 Butyl 12 ZYC-24 Me H ═O 0.510.52 13 ZYC-18 sec-But-2- H ═O 0.52 0.90 enyl 14 ZYC-16 H sec- ═O 0.660.70 But-2- enyl 15 17-β H H β-OH 2.21 3.01 Estradiol 16 Estrone H H ═O3.03 Not Done 17 17 α- H H α-OH 3.10 16.12 Estradiol

TABLE 2

Steroid Steroid concentration concentration needed to needed to protect50% protect 50% of neurons of neurons killed by 10 killed by 20 Com- mMGlutamate mM Glutamate pound R₁ R_(x) R_(z) ED₅₀ (μM) ED₅₀ (μM) 1 ZYC-33Adamantyl H α-OH 0.065 0.019 2 ZYC-34 t-Butyl H α-OH 0.24 0.26 3 Ent- HH α-OH 1.07 1.27 (17 β)- Estradiol

In view of the above, it will be seen that the several objects of theinvention are achieved. As various changes could be made in the aboveprocess and compound without departing from the scope of the invention,it is intended that all matter contained in the above description beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A process for treating an ophthalmologic diseaseselected from the group consisting of macular degeneration, retinaldegeneration, cataracts and glaucoma, the process comprisingadministering to a subject suffering from said ophthalmologic diseaseand in need thereof an effective dose of a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ is adamantyland R^(x) is hydrogen, methyl or methylpropyl.
 2. The process of claim 1wherein the compound has the formula:

or the enantiomer thereof.
 3. The process of claim 1 wherein thecompound has the formula:

or the enantiomer thereof.
 4. The process of claim 1 wherein theophthalmologic disease is retinal degeneration.
 5. The process of claim1 wherein the ophthalmologic disease is glaucoma.
 6. The process ofclaim 2 wherein the ophthalmologic disease is retinal degeneration. 7.The process of claim 2 wherein the ophthalmologic disease is maculardegeneration.